The DNA segment which contains the origin of mtDNA replication in Drosophila virilis has been cloned in E. coli and its nucleotide sequence has been determined. Using either native mtDNA or the chimeric DNA containing the origin of DNA replication, an in vitro mtDNA replication system has been developed from mitochondria isolated from Drosophila virilis embryos. A mutant which posesses an altered DNA polymerase alpha has been isolated from Drosophila melanogaster Schneider cell line #2 using aphidicolin as a selective reagent. An in vitro 2 microns DNA replication system has been developed from a crude extract of the yeast Saccharomyces cerevisiae. The origin and direction of 2 microns DNA replication have been determined both in vivo and in vitro.